Calcium agonists disrupt macrovascular and not microvascular cell barrier function, suggesting distinct responses to similar inflammatory mediators may represent a mechanism for targeting lung inflammation to specific vascular segments. Our prior work indicated that activation of store operated calcium entry by inflammatory calcium agonists inhibits adenylyl cyclase activity in pulmonary artery endothelial cells resulting in decreased cyclic AMP (cAMP) sufficient to increase permeability. Although microvascular endothelial cells (PMVECs) express a calcium inhibited isoform of adenylyl cyclase (AC6), calcium agonists neither decrease cAMP content nor disrupt the PMVEC barrier. Preliminary data indicate this calcium insensitivity is accompanied by a high rate of cAMP synthesis and turnover; when cAMP hydrolysis is disrupted in PMVECs calcium inhibits cAMP formation and inflammatory mediators induce intercellular gaps, demonstration AC6 critically regulates both PAEC and PMVEC barrier function. One cAMP-sensitive target that regulates endothelial cell permeability is membrane associated, on-erythroid spectrin. Disruption of spectrin binding to F-actin promotes intercellular gap formation in both PAECs spectrin. Disruption of spectrin binding to F-actin promotes intercellular gap formation in both PAECs and PMVECs, demonstrating spectrin's important role in regulating cell shape. Taken together our data support the idea that calcium inhibition of cAMP reduces the spectrin-F-actin association. In PAECs, whereas preservation of cAMP content promotes the spectrin-F-actin association in PMVECs. Thus, this proposal test the overall Hypothesis that calcium inhibition of cAMP formation decreases spectrin binding to F-actin important to increase lung endothelial cell permeability. Specific Aims test the related Hypotheses that: [1] cAMP accumulation regulates calcium inhibition of AC6 in PMVECs; [2] non-erythroid spectrin from F-actin increases macro- and microvascular endothelial cell permeability. Completion of this work will be important to further our understanding of the key signaling events that link calcium agonists to endothelial barrier disruption, a cardinal feature of multiple inflammatory lung diseases including acute respiratory distress syndrome, asthma, and reperfusion pulmonary edema.